1. Field of the Invention
This invention relates to cryo-fixation apparatus and method for the ultrarapid cooling of biological samples. Ultrarapid cooling is a preparatory step to the cryo preparation of biological samples in apparatus such as that described in commonly assigned U.S. Pat. Nos. 4,510,169 and 4,567,847 issued to John G. Linner and commonly referred to as "The Linner Process" or "The Linner Apparatus". It is well known in the medical arts that to examine biological samples and determine the cellular structure and function thereof, the samples must be "fixed" with minimal alteration of ultrastructural integrity prior to the application of nearly all analytical methodologies. The apparatus of this invention can be used to ultrarapidly cool biological samples without the formation of resolvable ice crystals so that the ultrastructural integrity of the sample is not altered. In samples of a biological material that are more than a few microns in thickness the ice crystals will usually range in size from amorphous at the leading edge to hexagonal in the interior of the tissue. There is a gradient effect during the formation of ice crystals.
The terms "biological material", "biological samples", "tissue samples", "tissue specimens", and "biological tissue" are used throughout this disclosure to refer to samples that can be ultrarapidly cooled by the method and apparatus of this invention. The terms are used interchangeably and are not intended as a limitation on the functional capability of the method or apparatus disclosed herein. The terms should be understood to include small tissue samples appropriate for microscopic examination and larger tissue masses such as corneas which are appropriate for transplantation. The terms should be understood to include any material composed of one or more cells, either individual or in complex with any matrix or in association with any chemical; and to include any biological or organic material and any cellular subportion, product or by-product thereof. The terms should be further understood to include without limitation sperm, eggs, embryos, blood components and other cellular components. The contemplated utility of the apparatus of this invention is not limited to specific types or sizes of tissue, rather it should be understood to refer to any biological material derived from cells (including prokaryotic, eukaryotic, plant or animal), viral particles and the like. The apparatus of this invention can be designed or adapted to any size, shape or type of cellular tissue. Therefore, the terms "tissue", "tissue specimen(s)" and "tissue sample(s)" are used interchangeably and are not limiting on the uses to which the method and apparatus of this invention can be placed.
Although the method and apparatus of this invention are preferably used as a preliminary step in the cryo-preparation of biological samples for ultrastructural analysis, i.e. electron microscopy, it should be understood that this is not intended as a limitation on the utility of the apparatus and method of this invention. To the contrary, the ultrarapid cooling method and apparatus of this invention have demonstrated utility in any area in which the ultrastructure of cellular components is desirably maintained in an unaltered state. Examples of such utility include, but are not limited to, electron microscopy, tissue preservation, tissue and organ transplants and various analytical and diagnostic methodologies. Therefore, although the method and apparatus of this invention are typically described in relationship to electron microscopy this should be understood not to be a limiting factor on the utility of the invention.
Although the examination of tissue by use of various microscopes or related magnifying apparatus has been practiced for many years, there has been an inherent problem in preparing tissue for use with contemporary high resolution analytical microscopes, such as the STEM electron microscopes, which permit the examination of sample constituents via X-ray analysis at powers of from 500.times. to 500,000.times. with point to point resolution of 2 to 3 Angstrom units. It is difficult to interpret the results of tissue analysis while concomitantly assessing the extent of various artifacts produced during the tissue preparation processes. It is thus essential that artifacts be avoided wherever possible. The term "artifact" refers to a product of artificial character due to extraneous agency. Another problem results from physical shrinkage of the tissue sample itself, which results in alteration of ultrastructure and massive rearrangement of infrastructural resolution.
During the so-called "Golden Age of Morphology" the predominant underlying goal in qualitative and quantitative microscopy has been an aesthetically pleasing image. This goal is readily attainable with the fixation methods and apparatus which are currently available. However, it has become essential that the aesthetically pleasing image, which is produced by the preparation process, also yield a tissue sample which accurately reflects the true condition of tissue in the living organism, i.e., approaching the "living state." This is the problem which is addressed and solved by the Linner.TM. Apparatus and Linner Process.TM.. One essential step in the preparation process is the cryopreparation or cryofixation procedure (as opposed to the freezing procedure). The cryo-preparation method and apparatus of this invention results in the preparation of tissue samples which are readily usable in known magnification and analytical apparatus.
In currently known cryopreparation and freeze-drying devices and methods, problems and limitations are encountered in attempts to rapidly cool the tissue sample without physically harming the sample. If the temperature decrease in the sample to its full depth does not take place at a sufficiently rapid rate, artifacts appear, the ultrastructural integrity of the sample may be damaged and the sample will not appear in its "living state". The prior art has therefore attempted to achieve a rapid rate of temperature decrease to the full depth of the sample, in order to minimize such damage.
Although the primary thrust of this application is in the preparation of tissue samples for analysis by current magnification apparatus, the invention is not intended to be so limited. More specifically, the "preparation" of tissue should be understood to refer to preparation of tissue for analysis as well as the cryofixation of tissue in anticipation of transplantation, modification, in vitro or in vivo cellular growth, fertilization, animated suspension or the more typical resin impregnation, setting, infiltration and analysis. The apparatus of this invention can be used to prepare tissue for any medical or analytical procedure without the ultrastructural damage previously thought to be inevitable in cryopreparation.
The apparatus of this invention is to be distinguished from contemporary freeze-drying apparatus. Freeze-drying is a technique which is well known in the art together with the equipment necessary to implement such freeze-drying. See, for example, U. S. Pat. No. 4,232,453. Although in certain freeze-drying techniques liquid nitrogen is used as a cooling medium, the tissue or sample itself does not attain such temperature. Freeze-drying normally contemplates sample temperatures of -50.degree. C. to -80.degree. C. In contrast, the ultrarapid cooling step of the cryopreparation process of the Linner Process.TM. and apparatus contemplate sample temperatures of -175.degree. C. or colder. Therefore, for purposes of this application the terms "cryopreparation" and "cryofixation" are used in distinction to conventional "freezing" technology.
2. Description of the Related Art
The most common prior art method for preparation of tissue samples for analysis is by means of chemical fixation and organic solvent dehydration. Inherent in prior art processes is the concomitant artifact creation, sample shrinkage and resultant damage to and modification of tissue characteristics. These tissue characteristic modifications, whether in the form of artifacts or the like, require interpretation by the individual or apparatus analyzing or evaluating the sample. This introduces, in many instances, an unsatisfactory risk of error. Chemical fixation is a well known technique and has served the analytical biologist well for many years and undoubtedly will continue to do so in certain limited applications. However, as the use of tissue sample analysis becomes more complex and the use of such analysis becomes more widespread, alternatives to chemical fixation are demanded. This is especially true as advances are being made in the magnification and analytical apparatus which are available. It is necessary that tissue preparation methods and the apparatus necessary to prepare tissue samples be equally advanced as the analytical tools, i.e., electron microscopes, which are being used to analyze the samples. Obviously, if the technology for tissue sample preparation is inferior to the technology of microscopy then the advanced microscopes cannot be used to full advantage by the morphologist or other tissue examiner.
Similarly, it is essential that cryopreparation methods and apparatus develop concurrently with other medical technology, i.e., surgical transplant techniques, bio-engineering and biogenetics. In short, cryo-preparation is an essential intermediate step in evolving processes using or analyzing cells or tissue. If cryo-preparation apparatus does not evolve then the thrust of medical technology into unexplained and unexplored medical arts will be blunted. The apparatus of this invention represents the cryopreparation breakthrough that will permit research into the use and preparation of biological tissue to keep pace with other advances in medical technology. The ultrarapid cooling apparatus of this invention provides the mechanism for eliminating the problems associated with available cryofixation apparatus.
An alternative to chemical fixation and organic solvent dehydration is freeze-drying cryofixed samples. Freeze-drying following cryofixation is a well documented and well known technique for tissue preservation. It has several advantages. Cryofixation results in a near-instantaneous arrest of cellular metabolism. There is also a stabilization and retention of soluble cell constituents through elimination of solvent contact with the sample. These are significant advantages to cryofixation freeze-drying that have resulted in a great deal of research in attempting to apply cryofixation and freezedrying techniques to known tissue preparation processes.
Unfortunately, freeze-drying technology inherently possesses a number of disadvantages relevant to tissue preparation methodologies. The primary disadvantage in currently available freezing techniques and apparatus is the inherent formation of ice crystals. As can be readily appreciated, the formation of ice crystals destroys the ultrastructural integrity of the tissue sample being reviewed. The image is distorted and the cytoplasm becomes reticulated. The formation of ice crystals in the sample can also result in a change in pH within microcompartments of the tissue (eutectic formation) which possibly can result in abnormal tertiary conformation of macromolecules. There is also the possibility that proteins will denature and precipitate. These are but a few of the disadvantages which are inherent in conventional freezing/freeze-drying processes.
This general topic is discussed in some detail together with other prior art methods in an article entitled "Freezing and Drying of Biological Tissues for Electron Microscopy", Louis Terracio and Karl G. Schwabe, published in The Journal of Histochemistry and Cytochemistry, Volume 29, No. 9 at pp. 1021-1028 (1981). Problems associated with artifact formation are described in "Understanding the Artefact Problem in Freeze-Fracture Replication: A Review", The Royal Microscopial Society, (1982) at pp. 103-123.
A general principle found applicable to freezing techniques, which has demonstrated utility in the preparation of tissue samples, is that as the cooling rate increases, tissue fluids can be "vitrified" without the separation of water to extracellular spaces. The term "vitrified" or "vitrification" refers to the cryopreparation of tissue samples without the formation of resolvable ice crystals within the cellular structure. It has been postulated that regardless of the rate of cooling, ice crystals may still be formed, but as the cooling rates increase the size of the intracellular ice crystals decreases. The small size or absence of ice crystals at high freeze rates is of course a substantial advantage in morphology retention as this results in minimal artifact creation and minimal ultrastructural alteration or damage during tissue dehydration. The apparatus of this invention provides the ultrarapid cooling of one or more tissue samples to the vitreous phase in less than one second. The ultrarapid cooling according to the present invention is followed by dehydration of the tissue sample while in the state of reduced partial pressure of water vapor without substantial ultrastructural damage to the tissue cells.
Historically, the criteria by which the techniques for rapid supercooling have been judged was not the cooling rate of the system but simply the temperature of the environment in which the tissue was frozen. Thus, the term rapid supercooling has been applied to any system in which the supercooling agent has a temperature of -150.degree. C. or below. The effectiveness of a cooling system, however, is dependent upon the rate at which heat is removed from the sample. Heat transfer is dependent not only on the temperature of the freezing system but also on its physical and thermal characteristics, as well as the size and thermal characteristics of the tissue.
The most commonly used technique for rapid super-cooling is to immerse or "quench" the sample in a fluid cooling bath. The most commonly used fluids for quenching are liquid nitrogen, isopentane, propane and fluorocarbons such as Freon 12 and Freon 22. Although liquid nitrogen is generally regarded as an ideal quenching fluid due to its low temperature (-196.degree. C.), there are inherent disadvantages in the use of liquid nitrogen due to the occurrence of tissue surface film boiling caused at least in part by the low heat of vaporization of liquid nitrogen. Film boiling is a characteristic of liquid nitrogen that inhibits the heat transfer rate by actually insulating the sample.
An alternative method for rapid supercooling is applying the tissue sample to the polished surface of a cryogenically cooled material such as the surface of a chilled metal block. This typically involves opposing the tissue sample to a polished flat metal surface by pressing it firmly against the surface of the metal. Silver and copper are typically used as the polished metal blocks. This method is designed to take advantage of the high thermal conductivities and heat capacities of these metals when cooled to liquid nitrogen or liquid helium temperatures. The critical step in chilling on the surface of a metal is making firm contact with the dry, chilled metal surface with no rotational, translational or rebounding motion. Certain commercially available apparatus having known utility in the medical arts address and provide "bounce-free" freezing. Credit for the development of this apparatus is generally accorded to Dr. Alan Boyne of the University of Maryland School of Medicine.
The Boyne apparatus and method included one or more copper bars partially submerged in a container filled with liquid nitrogen at -196.degree. C. The end of the copper bar was a mirror-finished smooth cryogenic surface, and the thermal conductivity of copper enabled the surface to be cooled. Cold nitrogen gas from vaporization of the liquid nitrogen, which escaped past the end of the copper bar, helped to reduce the contaminants on the cryogenic surface. A tissue sample was then dropped by gravity against the surface. To reduce the bounce of the sample against the surface, the Boyne sample delivery assembly employed a weight dampening system utilizing glycerol to absorb the impact. Each copper bar must be cleaned after slamming a sample. The drawbacks of the Boyne apparatus included problems of hydrocarbon contamination and condensation on the cryogenic surface, inability to eliminate all bounce or vibration between the sample and surface, undesirable precooling of the sample with escaping nitrogen gas, and delays for cleaning and regenerating the cryogenic surface of the copper bars between each sequential sample. The Boyne method and apparatus thus could not reliably provide tissue samples with good ice crystal-free zones nor was it capable of properly vitrifying the samples beyond a depth of 10 to 15 microns.
Further development of freezing tissue samples against a metal block has been credited to Jacques Escaig of Paris, France. The method and apparatus of Escaig is described in "Control of Different Parameters For Optimal Freezing Conditions", Jacques Escaig, published in Science of Biological Specimen Preparation, at pp. 117-122 (1984). The Escaig apparatus also is disclosed in Swiss Patent No. 614,532, French Patent No. 2,337,878 and German Patent No. 2,700,196. Escaig provided several significant features not shown in earlier methods or devices for vitrifying tissue against a metal block. The Escaig method and apparatus cool a copper block with liquid helium, rather than liquid nitrogen, in order to increase the cooling rate of a tissue sample or specimen. Escaig disclosed that the average thickness of ice crystal-free zones in the tissue sample were much larger when the copper block was cooled by liquid helium rather than liquid nitrogen. Additionally, Escaig pointed out that the factors influencing the freezing process, independent of the tissue sample itself, are thermal contact between the specimen and metal block, condition of the block surface, specimen slipping, sample holder bounce and sample holder contact strength. Escaig obtained some control of these factors by utilizing a vacuum pump to keep the metal block under vacuum of 1.times.10.sup.-3 mbar until just before slamming the specimen against the block, in order to reduce contamination on the block surface. Escaig also utilized an electromagnet to bring the specimen in contact with the block in order to improve mechanical contact between the specimen and the block.
According to the Escaig apparatus and method, a copper block is enclosed in a vacuum chamber. The chamber is then evacuated to approximately 1.times.10.sup.-3 mbar with an external vacuum pump system. After a vacuum is reached in the chamber, Escaig employed an external nonreusable cryogen source--liquid helium pumped from a reservoir--to cool the copper block. The liquid helium is transmitted to a passage adjacent to the block through a conduit in the vacuum chamber. When the block is cooled to the desired temperature, Escaig used an electropneumatic system to open a stem insert in the liquid helium passage for releasing cold helium gas for several seconds into the vacuum chamber itself. The cold helium gas admitted into the vacuum chamber is obtained from vaporization of the liquid helium which was used to cool the metal block. The cold helium gas raises the pressure inside the vacuum chamber. When atmospheric pressure is reached inside the vacuum chamber, a shutter providing access to the vacuum chamber is spring biased to open the chamber and activate downward movement of a sample delivery assembly, which plunges or slams the tissue sample through the shutter opening and against the block. Opening of the shutter also triggers closing of the stem insert to stop the release of cold helium gas into the chamber.
Several problems have been encountered in the Escaig apparatus. Because Escaig used cold helium gas to bring the vacuum chamber up to atmospheric pressure, a tissue sample precools for approximately 15 milliseconds as it descends through a layer of cold helium gas at atmospheric pressure prior to plunging or slamming against the metal block. The precooling effect of the Escaig device is undesirable due to unwanted effects, such as ice crystal formation, on the physiology of the tissue sample. Additionally, movement of liquid helium and helium gas through the vacuum chamber inevitably resulted in vibration of the metal block, which was undesirable because it reduced or prevented good mechanical contact between the specimen and block.
Escaig's use of nonreusable liquid helium to cool the block further has proved to be expensive, somewhat unsafe and cumbersome due to the necessity of recooling the entire liquid helium system between each tissue sample. The end result was extremely slow turnover time for regenerating the copper block between each sample. Additionally, cleaning the block in the Escaig apparatus proved difficult because of condensation on the block forming after a sample was slammed against the block. To remove the condensation, pressurized nitrogen gas and hot air could be applied against the block. However, removal of the block for repolishing or other cleaning required disassembly of the vacuum chamber itself. Even if the block was not removed, the problems in regenerating the block surface resulted in turnover time between samples which is commercially unacceptable. It was therefore not possible to use the Escaig device if a large number of sequential samples was desired.
The cryopreparation apparatus and method according to the present invention solves the problems inherent in the prior art including the Escaig apparatus and method. The present invention addresses the problems in the prior art of slow turnover time between the vitrification of tissue samples, by enabling several samples to be vitrified sequentially. The present invention also solves the problems in the prior art caused by undesirable precooling of the tissue sample or specimen before contact with the cryogenic block, cleaning and reheating the cryogenic block between each sample, and removal of the cryogenic block from the apparatus.